Lipases
The lipases comprise a family of enzymes with the capacity to catalyze hydrolysis of compounds including phospholipids, mono-, di-, and triglycerides, and acyl-coa thioesters. Lipases play important roles in lipid digestion and metabolism. Different lipases are distinguished by their substrate specificity, tissue distribution and subcellular localization.
Lipases have an important role in digestion. Triglycerides make up the predominant type of lipid in the human diet. Prior to absorption in the small intestine, triglycerides are broken down to monoglycerides and free fatty acids to allow solubilization and emulsification before micelle formation in conjunction with bile acids and phospholipids secreted by the liver. Secreted lipases that act within the lumen include lingual, gastric and pancreatic lipases, each having the ability to act under appropriate pH conditions. Modulating the activity of these enzymes has the potential to alter the processing and absorption of dietary fats. This may be important in the treatment of obesity or malabsorption syndromes such as those that occur in the presence of pancreatic insufficiency.
Lipases have an important role in lipid transport and lipoprotein metabolism. Subsequent to absorption across the intestinal mucosa, fatty acids are transported in complexes with cholesterol and protein molecules termed apoliporoteins. These complexes include particles known as chylomicrons, very low density lipoproteins ("VLDLs"), low density lipoproteins ("LDLs") and high density lipoproteins ("HDLs") depending upon their particular forms. Lipoprotein lipase and hepatic lipase are bound to act at the endothelial surfaces of extrahepatic and hepatic tissues, respectively. Deficiencies of these enzymes are associated with pathological levels of circulating lipoprotein particles. Lipoprotein lipase functions as a homodimer and has the dual functions of triglyceride hydrolase and ligand/bridging factor for receptor-mediated lipoprotein uptake. Severe mutations that cause LPL deficiency result in type I hyperlipoproteinemia, while less extreme mutations in LPL are linked to many disorders of lipoprotein metabolism.
Lipases have an important role in lipolysis. Free fatty acids derived from adipose tissue triglycerides are the most important fuel in mammals, providing more than half the caloric needs during fasting. The enzyme hormone-sensitive lipase plays a vital role in the mobilization of free fatty acids from adipose tissue by controlling the rate of lipolysis of stored triglycerides. Hormone sensitive lipase is activated by catecholamines through cyclic AMP-mediated phosphorylation of serine-563. Dephosphorylation is induced by insulin. While mice with homozygous-null mutations of their hormone-sensitive lipase genes induced by homologous recombination have been shown to enlarged adipocytes in their brown adipose tissue and to a lesser extent their white adipose tissue, they are not obese. White adipose tissue from homozygous null mice retain 40% of their wild type triacylglycerol lipase activity suggesting that one or more other, as yet uncharacterized, enzymes also mediate the hydrolysis of triglycerides stored in adipocytes. Hormone-sensitive lipase does not show sequence homology to the other characterized mammalian lipase proteins.
As identified above and in the cited references, lipase proteins are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of the lipase family of proteins. The present invention advances the state of the art by providing previously unidentified human proteins that have homology to known members of the lipase family of proteins.
The present invention has a substantial similarity to microsomal arylacetamide deacetylase (DAC). DAC Full-length cDNA was isolated from a human liver lambda gt 11 library. This clone encodes an open reading frame of 400 amino acids with a deduced molecular mass of 45.7 kDa and contains two putative glycosylation sites. The 3'-untranslated region contains two putative polyadenylation signals competes against the activity of cytosolic arylamine N-acetyltransferase, which catalyzes one of the initial biotransformation pathways for arylamine and heterocyclic amine carcinogens in many species and tissues. Two extended regions of significant sequence homology with hormone-sensitive lipase and with lipase 2 from Moraxella TA144 were identified.
Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that functions as the rate-limiting enzyme for the mobilization of free fatty acids in adipose tissue. HSL-derived fatty acids are bound by ALBP to facilitate intracellular trafficking of hydrophobic lipids. HSL has broad substrate specificity; in addition to triacylglycerol, HSL can also catalyze the hydrolysis of diacylglycerol, 1 monoacylglycerol, cholesteryl esters, lipoidal esters of steroid hormones, and retinyl esters in adipose tissue. In the rat, HSL is a 767-aa protein that has a molecular mass of 84 kDa on SDS/PAGE. Its primary sequence is unrelated to any of the other known mammalian lipases; however, within its catalytic domain, it shares some sequence similarity with liver arylacetamide deacetylase as well as with esterases isolated from several bacteria. The secondary structure of the C-terminal portion of HSL has been demonstrated to possess homology with some features of the secondary structure of acetylcholinesterase and of fungal lipases from Geotrichum candidum and Candida rugosa, whereas the N-terminal 320 aa have no primary or secondary structural similarity with any known proteins. The activity of HSL against triacylglycerol and cholesteryl ester substrates is regulated acutely via phosphorylation-dephosphorylation reactions. The activation of HSL by fast-acting lipolytic hormones (catecholamines, glucagon, corticotropin) involves a hormone/receptor-induced increase in the cellular concentration of cAMP, which then activates cAMP-dependent protein kinase, resulting in the phosphorylation of HSL and an increase in hydrolytic activity.
Activity determination and immunoblot analysis of DAC in human target tissues for arylamine carcinogens revealed that in extrahepatic tissues, additional enzymes are responsible for any deacetylation activity, whereas a single enzyme predominantly catalyzes this hydrolytic reaction in liver.
The present invention also provide an important tool to study deacetylation and its effects on the metabolic activation of arylamine and heterocyclic amine carcinogens. For a review related to the present invention, see Shen et al., PNAS Vol. 96, Issue 10, 5528-5532, May 11, 1999; Probst et al., J Biol Chem Aug. 26, 1994;269(34):21650-6; Choo et al., (1998). Appl. Environ. Microbiol. 64: 486-491.
Lipase proteins, particularly members of the hormone-sensitive lipase(arylacetamide deacetylase) subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of lipase proteins. The present invention advances the state of the art by providing a previously unidentified human lipase proteins that have homology to members of the hormone-sensitive lipase(arylacetamide deacetylase) subfamily.